Interactions of mononuclear leukocytes with vascular endothelium plays a pivotal role in acute ana chronic inflammation, immune reactions, vascular injury and atherosclerosis. In the previous funding period, four major accomplishments occurred. (1) We characterized novel adhesion pathways, one in endothelium and one in T cells subsets and one in B cells. (2) We developed a sensitive flow assay to analyze chemokine effects on leukocytes. (3) We determined adhesion pathways responsible for T cell subset recruitment using in vitro flow assays and by employing a murine adoptive transfer protocol. (4) We established the "blot rolling" assay to identify and characterize T cell and endothelial cell ligands for selectins. This proposal builds on this scientific progress and will consist of four, interrelated specific aims that will focus on the role of selectin mediated adhesion in mononuclear leukocyte recruitment using in vivo and in vitro models. In Specific Aim 1, we will define and biochemically characterize the inducible T cell ligands for E- and P-selectins. The experimental approach will utilize a combination of a newly developed "blot rolling" assay and a traditional affinity isolation. In Specific Aim 2, we will define and biochemically characterize the inducible L-selectin ligands on peripheral vascular endothelium using the "blot rolling" assay described in Specific Aim 1. In Specific Aim 3, we will determine the influence of an initial "wave" of leukocyte trafficking on a second "wave" of leukocytes. Experimental models of inflammation demonstrate that leukocyte recruitment is focal and often exhibits a temporal pattern of leukocyte subset recruitment, with neutrophils accumulating first followed by accumulation of mononuclear leukocytes (monocytes and lymphocytes). The proposed studies will test our working hypothesis that the first wave of adherent leukocyte alters the vascular endothelium with respect to its ability to support adhesive interactions and emigration of a second wave of leukocytes. In Specific Aim 4 we will test the hypothesis that different vascular tissues undergoing immune reactions support differential recruitment of T cells subsets. We will examine whether an antigen-specific allergic reaction in the gut selectively recruits adoptively transferred T cell subsets. The studies will employ immunohistochemistry and intravital microscopy techniques to quantify T cell recruitment. The information gained from these studies will help delineate the mechanisms underlying T cell subset interactions with vascular endothelium and may thus define new targets for therapeutic intervention in the treatment of immune and inflammatory diseases.